Expanded vacuum-stable gels for multiplexed high-resolution spatial histopathology

Cellular organization and functions encompass multiple scales in vivo. Emerging high-plex imaging technologies are limited in resolving subcellular biomolecular features. Expansion Microscopy (ExM) and related techniques physically expand samples for enhanced spatial resolution, but are challenging to be combined with high-plex imaging technologies to enable integrative multiscaled tissue biology insights. Here, we introduce Expand and comPRESS hydrOgels (ExPRESSO), an ExM framework that allows high-plex protein staining, physical expansion, and removal of water, while retaining the lateral tissue expansion. We demonstrate ExPRESSO imaging of archival clinical tissue samples on Multiplexed Ion Beam Imaging and Imaging Mass Cytometry platforms, with detection capabilities of > 40 markers. Application of ExPRESSO on archival human lymphoid and brain tissues resolved tissue architecture at the subcellular level, particularly that of the blood-brain barrier. ExPRESSO hence provides a platform for extending the analysis compatibility of hydrogel-expanded biospecimens to mass spectrometry, with minimal modifications to protocols and instrumentation.

This manuscript has been previously reviewed at another journal that is not operating a transparent peer review scheme. This document only contains reviewer comments and rebuttal letters for versions considered at Nature Communications.
Bai et al have presented a revised paper on a novel approach for expansion imaging that involves spatially-resolved mass analysis. The authors have demonstrated a commendable level of responsiveness by providing thorough and detailed responses to the multiple requests made by the reviewers and editors. While it may be a minor detail, I still hold a different view from the authors with regards to the reversibility of crosslinks through the use of a detergent. Although the authors have made efforts to find publications demonstrating the contrary, covalent bonds between proteins are generally difficult to reverse due to the strong and stable bond created by the sharing of electrons between atoms. Breaking a covalent bond typically requires a significant amount of energy, which makes it challenging to reverse covalent bonds formed through crosslinking with paraformaldehyde or other crosslinking agents. Nonetheless, I appreciate the authors' perspective on this matter and commend their efforts to address it in the paper. My major concern is, as Reviewer 3, I remain unconvinced that the EXPRESSO technique is suitable for depth profiling and 3D reconstruction. The authors have not provided sufficient evidence to demonstrate the following: -Homogeneity in the expansion process: The authors need to show that the expansion of tissue is sufficiently homogeneous across the sample, as non-uniform expansion could lead to errors in depth profiling and 3D reconstruction.
-Absence of background noise: It is important to ensure that the compression in the Z plane does not result in the fusion or superposition of certain structures, which could create background noise in the images. At this spatial resolution, it could be a critical point as in the case of fluorescence microscopy, where confocal analysis is necessary to obtain the signal from a single plane to ensure that the signal is specific to the region of interest.
-Homogeneous depth ablation: The authors need to demonstrate that the depth ablation is homogeneous across different tissue structures. The presence of signal loss may indicate uneven ablation, which could lead to inaccuracies in the 3D reconstruction. By addressing these concerns, the authors can provide a more comprehensive assessment of the suitability of EXPRESSO for depth profiling and 3D reconstruction. I would advise the authors to consider removing this section, as it is not the main focus of the paper, and instead focus on addressing these points in a separate publication.
The last point to consider is that interferences may arise due to the presence of the gel. As demonstrated in Figure R2 on Page 24, the authors noted signal losses, but were unable to provide a precise explanation. This effect is also observed in other mass spectrometry imaging techniques, such as SIMS or MALDI-axialTOF. These signal losses represent an extreme manifestation of an ionization problem that results from the diversity of structures. This phenomenon can be less pronounced, leading to differences in signal intensity during analysis, independent of the protein concentration. Consequently, I remain skeptical about the quantitative accuracy of the results.

Reviewer #2 (Remarks to the Author)
In their rebuttal/cover letter, the authors address the two major points I raised in the second round of prior review.
1. With respect to signal intensity after expansion, they comment on various aspects of the protocol that may increase access of antibodies to epitopes and limit the predicted loss of signal due to volume dilution upon expansion. These are reasonable and may be correct, but at this point the key is to make the reader very aware of this matter because if the epitopes of biological concern are among those w=here signal is substantially lost, then this becomes a severe limiting feature of the method. Perusing the list provided by the authors as Table R1, many although not all of the data fit into a pretty clear pattern. Staining for epitopes associated with structural elements such as nucleic acids and extracellular matrix show less than predicted loss of signal and even a gain relative to non-ExPRESSO, whereas those associated with membrane proteins (CD3 being a major exception), lose signal by several fold. This makes sense as the epitopes on macromolecular repetitious structures like matrix and nucleic acids would benefit from 'unpacking' whereas sparse membrane proteins would not and they would suffer from dilution as expected, albeit less than theoretically maximal. I think this table should be incorporated into the final manuscript and the authors should include a discussion on the issue citing to these data, which can provide a guide to users about whether the markers of interest in their study are or are not likely to loss signal when the sample is processed by ExPRESSO and they can then take into account S/N loss vs. resolution gain in determining whether to adopt the method. 2. With respect to which antibodies work and which do not, it is obviously not possible or practical for the authors to provide a comprehensive guide to users on this issue, as the basis for failure in this method vs. others is not trivially apparent. Relying on information in the citations, the accumulating database on antibodies for MIBI use, and direct in lab testing will be how users can most efficiently proceed when establishing a panel for a specific purpose.
With the changes I suggested in point 1, I can recommend this paper to NC, pending satisfactory responses to the points raised by other referees.

Reviewer #3 (Remarks to the Author)
The reviewer does not have any further concerns regarding the current version of the manuscript, which has been adequately addressed and is now suitable for publication in Nature Communications.